Interaction of actin and myosin is the basis of muscle contraction and force generation in muscle, as well as the basis of cellular motion and intracellular traffic. In myosin II, actin binding initiates the powerstroke, a single force-generating event, when myosin changes its conformation and propels actin filament relative to thick filament in muscle or transfers cargo along actin cytoskeleton in cells. Acto-myosin interaction modulates myosin ATPase activity, enhancing the rate of ATP hydrolysis products release. The goal of this project is to determine the mechanism of myosin ATPase actin activation. We hypothesize that the powerstroke is regulated by actin via modulation of the relay loop - relay helix interaction in the force generating region of myosin. Actin binding changes the relay loop conformation and destabilizes the relay helix in the pre- powerstroke, inducing the powerstroke. We will measure the conformation of the relay helix during myosin- ATP and myosin-actin interaction, using newly developed assay, based on site-directed labeling with fluorescent and spin probes, pulsed electron paramagnetic resonance and transient time-resolved fluorimetry, two complementary high-resolution spectroscopic techniques. As we have shown previously these mutations and labeling do not affect myosin function. We will introduce functional mutations into the relay loop to perturb actin activation, and study activation mechanism in detail, analyzing myosin conformation and timing of this conformational change during actin activation. There are two Aims: Aim 1. Technology development to study actin activation of force production in myosin. This is a technical basis to achieve our goal. We will use our previously developed approach to measure kinetics of the relay helix conformational change. We will upgrade our current transient time-resolved fluorimeter with 3 syringe/2mixer stopped flow apparatus. First, myosin will be prepared in the transient pre-powerstroke state by rapid mixing with ATP. Then we will rapidly mix prepared myosin with actin to initiate acto-myosin interaction. The kinetics of actin activated powerstroke will be monitored via conformational change of the relay helix by transient time- resolved fluorimetry. We will answer the question: what is the timing of the force generation and how is it related to nucleotide binding, and products of hydrolysis release? Aim 2. The mechanism of myosin ATPase actin activation. The relay helix conformation and its transient structural dynamics in functional myosin mutants will be studied using pulsed electron paramagnetic resonance and transient time-resolved fluorimetry. We will answer two questions: is the relay loop a regulating element in actin activation of myosin? What is the mechanism of this activation? This study is of fundamental importance for understanding the mechanism of muscle function and muscle malfunction on submolecular level. Knowledge of these mechanisms will allow rational design of muscle malfunction treatment, as well as better understanding of the mechanism of muscle contraction. PUBLIC HEALTH RELEVANCE: The ultimate goal of the project is to understand how the force is generated in muscle. In the proposed research, we will study how myosin, a molecular motor, is activated by actin for the force production. To reach the goal, we will combine molecular biology, biochemistry and biophysical spectroscopy. This study is of fundamental importance for understanding the mechanism of muscle function and muscle malfunction on submolecular level. Knowledge of this mechanism will allow rational design of muscle malfunction treatment, as well as better understanding of the mechanism of muscle contraction.